Primer probe composition and kit for detecting brain infection pathogens and preparation method of primer probe composition

Abstract

The invention provides a primer probe composition for detecting brain infection pathogens, a kit and a preparation method of the kit, and belongs to the technical field of biological detection. The primer probe composition is used for detecting chikungunya virus, Sween encephalitis virus, Japanese encephalitis virus, forest encephalitis virus, American East equine encephalitis virus, American West equine encephalitis virus, West Nile virus, echovirus, Coxsackie virus, cytomegalovirus, herpes simplex virus, varicella-zoster virus and parotitis virus. The kit comprises an upstream primer, a downstream primer and a fluorescent probe, wherein the upstream primer, the downstream primer and the fluorescent probe are designed for schistosoma and ribonuclease P, and the upstream primer, the downstream primer and the fluorescent probe are designed for streptococcus agalactiae, escherichia coli, listeria monocytogenes, neisseria meningitidis, streptococcus pneumoniae and pork tapeworm; the method has the advantages of simple operation steps, short detection period, high efficiency, low cost, accurate result, avoidance of false positive phenomenon, high detection sensitivity, strong specificity, and realization of the possibility of multi-channel detection in a single-tube system.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular relates to a primer-probe combination, a kit and a preparation method thereof for detecting pathogens of brain infection. Background Art

[0002] Brain infections may be caused by viruses, bacteria, fungi, or protozoa or parasites. Symptoms include fever, headache, and altered mental status, often with seizures or focal neurologic deficits. Infections can cause inflammation of the brain (encephalitis). Viruses are the most common cause of encephalitis. Infections can also cause inflammation of the layers of tissue covering the brain and spinal cord (meninges), called meningitis. Meningitis can be caused by bacteria, viruses, fungi, noninfectious disorders, or drugs. Encephalitis is caused by direct invasion of the brain by viruses that cause primary encephalitis. These infections may be epidemic (such as arboviruses, echoviruses, coxsackieviruses) and sporadic (such as encephalitis caused by cytomegalovirus, herpes simplex virus, varicella-zoster virus, or mumps virus). In newborns and young infants, the most common causes of bacterial meningitis are group B Streptococcus (especially S. agalactiae), Escherichia coli, and Listeria. In older infants, children, and young adults, the most common causes are Neisseria meningitidis (meningococci) and Streptococcus pneumoniae (pneumococci). In middle-aged and older people, the most common cause is S. pneumoniae. Neurocysticercosis is caused by the larvae of the pork tapeworm, and schistosomiasis is an infection caused by schistosomes.

[0003] Encephalitis caused by mosquito-borne arboviruses is usually more common in spring, summer and early autumn when the climate is warmer. Arboviruses that cause encephalitis include Chikungunya virus, St. Louis encephalitis virus, Japanese encephalitis virus, forest encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, and West Nile virus. Children are more common in patients, and most cases are clustered. Chikungunya virus (CHIKV) is an acute infectious pathogen that causes chikungunya fever. It is a zoonotic disease that is mainly transmitted by infected Aedes mosquitoes. The genome is a single-stranded positive-strand RNA, belonging to the genus Alphavirus of the family Togaviridae. Clinical symptoms are acute fever, rash and chronic polyarthritis. St.Louts encephalitis virus (StLEV) is an acute mosquito-borne virus that causes St.Louis encephalitis. It is a small (40-60nm), enveloped, positive-sense, single-stranded RNA virus that replicates in the cytoplasm of infected cells. Clinical features include inflammation and damage to the meninges and brain parenchyma. Japanese encephalitis virus (JEV), also known as Japanese encephalitis virus, is the pathogen that causes epidemic Japanese encephalitis. It is an enveloped, single-stranded, positive-sense RNA virus. It is a zoonotic disease that causes central nervous system diseases in humans and reproductive disorders in pigs. The disease mainly affects children, with clinical manifestations of high fever, convulsions, impaired consciousness, pathological reflexes and meningeal irritation signs. Severe cases can lead to death or post-healing neurological sequelae. Tick-borne encephalitis virus (TBEV), also known as tick-borne encephalitis virus, can cause natural epidemic diseases in forest areas. Its genome is a single-stranded positive-strand RNA of about 11kb. The virus mainly invades the central nervous system. Clinical symptoms include fever, headache, impaired consciousness, meningeal irritation, etc., leaving paralytic sequelae, and in severe cases, death. Eastern equine encephalomyelitis virus (EEEV) is an acute zoonotic pathogen that causes equine encephalitis in the eastern United States. Its genome is a single-stranded positive-strand RNA. It can be transmitted not only through insect vectors, but also through aerosols. Clinical symptoms include high fever, severe headache, neck stiffness, vomiting, lethargy, coma, convulsions and other encephalitis symptoms. Western equine encephalitis virus (WEEV) is an acute zoonotic pathogen that causes equine encephalitis in the western United States. It is a single-stranded positive-strand RNA that is mainly transmitted by mosquitoes. Clinical symptoms include fever, headache, malaise, vomiting, and neck stiffness.West Nile virus (WNV) is a zoonotic pathogen that can cause West Nile encephalitis. It is an enveloped, single-stranded, positive-sense RNA virus. Infection with the virus can cause encephalitis symptoms such as fever, headache, rash, swollen lymph nodes, convulsions, and impaired consciousness.

[0004] Echovirus (ECHO) belongs to enterovirus, which is composed of a simple capsid and single-stranded positive-strand RNA. It is mainly transmitted through fecal-oral or oral-oral transmission, and can cause central nervous system complications such as acute fever, gastrointestinal and respiratory discomfort, encephalitis, myocarditis, and conjunctivitis. Coxsackievirus (CV) is a pathogen that causes serious diseases such as aseptic meningitis, viral myocarditis, dilated cardiomyopathy, hepatitis, and pancreatitis. It is a single-stranded positive-strand RNA virus. The pathogenic Coxsackievirus mainly includes Coxsackievirus A group 6, Coxsackievirus A group 10, and Coxsackievirus A group 16. The clinical symptoms are fever, cold, mild upper respiratory symptoms, diarrhea, and gastrointestinal diseases.

[0005] Cytomegalovirus (CMV) is a double-stranded DNA virus with a typical herpes virus morphology. It is also called cell inclusion virus because the cells it infects swell and become round, the nucleus becomes larger, and huge eosinophilic inclusion bodies appear in the nucleus. The virus has strict species specificity for hosts and cultured cells. Human cytomegalovirus (HCMV) can only infect humans. It can not only be transmitted congenitally from mother to child, but also be transmitted acquired through saliva, breast milk, urine, surgery, organ transplantation and other medical sources. After being infected with cytomegalovirus, people will develop complications such as encephalitis, gastroenteritis, retinitis, hepatitis, pneumonia, etc. Since CMV can infect most cells and organs, its morbidity and mortality are higher than other herpes viruses. Herpes simplex virus (HSV) is a pathogen that seriously endangers human health and causes skin diseases and sexually transmitted diseases. Humans are its only natural host and are generally susceptible. The genome is linear double-stranded DNA. Based on the difference in antigenicity, the virus is divided into two serotypes: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). HSV-1 is generally transmitted through the mouth, droplets and contaminated hands, mainly causing gingivitis, keratitis, conjunctivitis, herpes labialis, encephalitis and eczema. HSV-2 is transmitted through sexual contact and vertical transmission from mother to child, mainly causing skin and mucous membrane infection in the genital area and neonatal infection, causing genital herpes. Varicella-zoster virus (VZV) is a double-stranded DNA virus. Humans are the only natural host of the virus. It is mainly transmitted through droplets, aerosols or contact, and is lymphotropic and neurotropic. When cellular immune function is low, latent varicella-zoster virus is activated, causing meningitis, encephalitis, cerebral vasculitis, myelitis, cerebellitis and Hunter syndrome, etc. Clinical symptoms include fever, headache, nausea and vomiting, mental and behavioral abnormalities, epileptic seizures, ataxia, stiff neck, etc. In severe cases, it can cause delirium, coma, respiratory or circulatory failure. Mumps virus (MuV) is a serious infectious pathogen. Humans are its only natural host, and air droplets are the main transmission route of the virus. In addition to causing mumps, MuV can also invade multiple organs and the central nervous system, leading to many clinical symptoms and complications such as encephalitis, meningitis, orchitis, myocarditis, pancreatitis, nephritis, etc. It can also cause long-term sequelae such as paralysis, epileptic seizures, cranial nerve palsy, hydrocephalus, and deafness.

[0006] Streptococcus agalactiae (GBS) is a Gram-positive streptococcus, an important cause of postpartum sepsis and neonatal meningitis in pregnant women. It parasitizes in the reproductive tract of pregnant women and can cause infection in infants. It can also cause postpartum infection, bacteremia, endocarditis, skin and soft tissue infection, and osteomyelitis. The bacteria can cause sepsis and meningitis in animals and has a high mortality rate. Escherichia coli (E. coli) is a conditional pathogen that can cause gastrointestinal infections or urethral and other local tissue and organ infections in humans and various animals under certain conditions. It can also cause urethral infections, arthritis, meningitis, and septicemic infections. Listeria monocytogenes (L.monocytogenes) is a zoonotic pathogen that is widely present in nature. It is one of the main pathogens that threaten human health in refrigerated foods. It can cause listeriosis in humans and livestock, and the main symptoms after infection are sepsis, meningitis and monocytosis. Neisseria meningitidis (NME) is a Gram-negative bacterium that only infects humans. Infection with this bacterium can cause meningitis and meningococcemia. Streptococcus pneumoniae (SPN) usually resides in the nasopharynx of normal people. When the body's resistance is reduced, it can often invade the lung tissue and cause pneumonia. In addition, it can also cause sepsis, sinusitis, otitis media and purulent meningitis. Typical symptoms are sudden chills, high fever, chest pain, cough and rust-colored sputum. The pork tapeworm (Taenia solium) is a zoonotic parasitic worm and the main human parasitic tapeworm in China. The intermediate host is mainly pigs, and humans are the only definitive host. The pork tapeworm larvae enter the blood and spread to all parts of the body, forming cysts that cause brain cysticercosis. The cysts rupture and spill their contents into the cerebrospinal fluid, causing meningitis. Schistosoma is a type of trematode that parasitizes in blood vessels. Schistosoma infection causes schistosomiasis, a zoonotic parasitic disease. Some patients with schistosomiasis will form cell clusters caused by inflammation in the brain. Clinical symptoms include epileptic seizures, headaches, personality changes, and mental disorders.

[0007] Brain infection-related pathogens pose a huge threat to human public health and the sustainable development of animal husbandry, and there is an urgent need for rapid, immediate and accurate diagnostic solutions. At present, the detection methods for encephalitis-related pathogens mainly include virus isolation and culture, serological detection and fluorescent quantitative PCR. The traditional virus isolation method is the gold standard for the detection and identification of encephalitis-related pathogens, but this method is complicated, time-consuming and labor-intensive, and has poor sensitivity, which makes it difficult to meet the needs of rapid diagnosis and treatment of diseases. The serological detection method has low sensitivity, poor specificity, and cumbersome operation. It can only detect one pathogen at a time. When detecting multiple items, a large number of serum samples need to be collected, which is not conducive to the timely diagnosis and treatment of the disease. The conventional PCR method requires gel electrophoresis to determine the detection results, which is labor-intensive and the product is prone to aerosol contamination. Summary of the invention

[0008] In view of the above technical defects, the present invention provides a primer-probe composition, a kit and a preparation method thereof for detecting pathogens of brain infection. The probe melting curve technology is adopted to realize multi-channel detection of different pathogens, which is rapid, accurate, has high sensitivity and detection throughput, and good specificity. It avoids the shortcomings of conventional PCR methods, such as cumbersome operation and easy occurrence of false positives, and is helpful to prevent and control the prevalence of various encephalitis-related pathogens.

[0009] The technical solution adopted by the present invention is: A primer-probe composition for detecting pathogens of brain infection, comprising: SEQ ID NO.1: an upstream primer designed for Chikungunya virus, the sequence of the upstream primer is 5'-ccaacgctatcgcttgattacatc-3'; referred to as CHIKV-F; SEQ ID NO.2: A downstream primer designed for Chikungunya virus, the sequence of which is 5'-atgggtagacgccggtgaag-3'; referred to as CHIKV-R; SEQ ID NO.3: A fluorescent probe designed for Chikungunya virus, the sequence of which is 5'-tgcgaatacaaaaccgtcatcccgtctccg-3'; referred to as CHIKV-P; SEQ ID NO.4: An upstream primer designed for St. Louis encephalitis virus, the sequence of which is 5'-aaccacccttcggcgactc-3'; StLEV-F for short; SEQ ID NO.5: A downstream primer designed for St. Louis encephalitis virus, the sequence of which is 5'-gccttcccaatgctgcttcc-3'; referred to as StLEV-R; SEQ ID NO.6: A fluorescent probe designed for St. Louis encephalitis virus, the sequence of which is 5'-tatcgtcgtcggaagaggcaccaccca-3'; StLEV-P for short; SEQ ID NO.7: An upstream primer designed for Japanese encephalitis virus, the sequence of which is 5'-tgacaacagaaggtgagaaagcc-3'; referred to as JEV-F; SEQ ID NO.8: A downstream primer designed for Japanese encephalitis virus, the sequence of which is 5'-gtggcctgacgttggtctttc-3'; referred to as JEV-R; SEQ ID NO.9: A fluorescent probe designed for Japanese encephalitis virus, the sequence of which is 5'-acttccagtgagcagggacctacttccgag-3'; referred to as JEV-P; SEQ ID NO.10: An upstream primer designed for forest encephalitis virus, the sequence of which is 5'-ctttcggacagcagagagtgttc-3'; referred to as TBEV-F; SEQ ID NO.11: A downstream primer designed for forest encephalitis virus, the sequence of which is 5'-gccagtcgttccaaaatccaatc-3'; referred to as TBEV-R; SEQ ID NO.12: A fluorescent probe designed for forest encephalitis virus, the sequence of which is 5'-agttgacccaaaggcacaggaacctcagc-3'; referred to as TBEV-P; SEQ ID NO.13: An upstream primer designed for Eastern Equine Encephalitis Virus of the United States, the sequence of the upstream primer is 5'-gtgtcaggtgtttacaggtgtttac-3'; referred to as EEEV-F; SEQ ID NO.14: A downstream primer designed for Eastern Equine Encephalitis Virus of the United States, the sequence of the downstream primer is 5'-catggtcgatagagcactcttcc-3'; referred to as EEEV-R; SEQ ID NO.15: A fluorescent probe designed for Eastern Equine Encephalitis Virus of the United States, the sequence of the fluorescent probe is 5'-cgctctacatacgcctcgctcatctggg-3'; referred to as EEEV-P; SEQ ID NO.16: An upstream primer designed for Western equine encephalitis virus of the United States, the sequence of the upstream primer is 5'-ccaccgccaaagaagaagaagag-3'; referred to as WEEV-F; SEQ ID NO.17: A downstream primer designed for Western Equine Encephalitis Virus of the United States, the sequence of the downstream primer is 5'-cgctgtcgtttccctggtttag-3'; referred to as WEEV-R; SEQ ID NO.18: A fluorescent probe designed for Western equine encephalitis virus of the United States, the sequence of which is 5'-cttgcgtttcgtcttcttggcttgttgcttct-3'; referred to as WEEV-P; SEQ ID NO.19: An upstream primer designed for West Nile virus, the sequence of the upstream primer is 5'-ggaggaacagagagacgttaatgg-3'; abbreviated as WNV-F; SEQ ID NO.20: A downstream primer designed for West Nile virus, the sequence of which is 5'-ccctcttgtgagcccaatgc-3'; abbreviated as WNV-R; SEQ ID NO.21: A fluorescent probe designed for West Nile virus, the sequence of which is 5'-tgaggaaccacacgccacgaagcagtc-3'; WNV-P for short; SEQ ID NO.22: An upstream primer designed for echovirus, the sequence of which is 5'-gcgtccctgaagcagaaatgg-3'; referred to as ECHO-F; SEQ ID NO.23: A downstream primer designed for echovirus, the sequence of which is 5'-cgaatccgaagtgaacacatgagg-3'; referred to as ECHO-R; SEQ ID NO.24: A fluorescent probe designed for echovirus, the sequence of which is 5'-tcctcctcggtggtcaactctgtggca-3'; referred to as ECHO-P; SEQ ID NO.25: An upstream primer designed for Coxsackievirus group A type 6, the sequence of the upstream primer is 5'-gctttgatgccgagttcac-3'; abbreviated as CVA6-F; SEQ ID NO.26: A downstream primer designed for Coxsackievirus group A type 6, the sequence of the downstream primer is 5'-gtctgccattgatatgatttcc-3'; abbreviated as CVA6-R; SEQ ID NO.27: A fluorescent probe designed for Coxsackievirus group A type 6, the sequence of which is 5'-ccaacctcaatgacagcacgacgc-3'; referred to as CVA6-P; SEQ ID NO.28: An upstream primer designed for Coxsackievirus group A type 10, the sequence of the upstream primer is 5'-ttgtgtggttaacagaaatggg-3'; abbreviated as CVA10-F; SEQ ID NO.29: A downstream primer designed for Coxsackievirus A group 10, the sequence of the downstream primer is 5'-ctgcgttgaacctcatgtatg-3'; referred to as CVA10-R; SEQ ID NO.30: A fluorescent probe designed for Coxsackievirus A group 10, the sequence of which is 5'-aaccactatcaaccacttcttctcccgc-3'; referred to as CVA10-P; SEQ ID NO.31: An upstream primer designed for Coxsackievirus group A type 16, the sequence of the upstream primer is 5'-acacaagagacagccattgg-3'; abbreviated as CVA16-F; SEQ ID NO.32: A downstream primer designed for Coxsackievirus group A type 16, the sequence of the downstream primer is 5'-tcagcatcaaagcgcatg-3'; referred to as CVA16-R; SEQ ID NO.33: A fluorescent probe designed for Coxsackievirus group A type 16, the sequence of which is 5'-aataactcgcatttccgccgcag-3'; referred to as CVA16-P; SEQ ID NO.34: An upstream primer designed for cytomegalovirus, the sequence of which is 5'-ttccccgaaggcaccagatg-3'; referred to as CMV-F; SEQ ID NO.35: A downstream primer designed for cytomegalovirus, the sequence of which is 5'-gggacctattcgttttcacacctac-3'; referred to as CMV-R; SEQ ID NO.36: A fluorescent probe designed for cytomegalovirus, the sequence of which is 5'-acgccgtgctcttcttcgactcgcc-3'; referred to as CMV-P; SEQ ID NO.37: An upstream primer designed for herpes simplex virus type I, the sequence of the upstream primer is 5'-tggagcaccgagccaagg-3'; referred to as HSV1-F; SEQ ID NO.38: A downstream primer designed for herpes simplex virus type I, the sequence of the downstream primer is 5'-cgatgctgtccaccgtcac-3'; referred to as HSV1-R; SEQ ID NO.39: A fluorescent probe designed for herpes simplex virus type I, the sequence of which is 5'-ctcctgtaagtacgccctcccgctgc-3'; referred to as HSV1-P; SEQ ID NO.40: An upstream primer designed for herpes simplex virus type II, the sequence of the upstream primer is 5'-caacttggcctcgtcaaaccc-3'; referred to as HSV2-F; SEQ ID NO.41: A downstream primer designed for herpes simplex virus type II, the sequence of the downstream primer is 5'-tgcaacgcaatcccatgaagg-3'; referred to as HSV2-R; SEQ ID NO.42: A fluorescent probe designed for herpes simplex virus type II, the sequence of which is 5'-ccgctcaccaccaaggaactcaagacttc-3'; referred to as HSV2-P; SEQ ID NO.43: An upstream primer designed for varicella-zoster virus, the sequence of the upstream primer is 5'-ctctggtgaagccgcacttac-3'; referred to as VZV-F; SEQ ID NO.44: A downstream primer designed for varicella-zoster virus, the sequence of which is 5'-cgaatgttggaaactgttggtcttc-3'; referred to as VZV-R; SEQ ID NO.45: A fluorescent probe designed for varicella-zoster virus, the sequence of which is 5'-ccgacccactctcccaaccctaacagc-3'; referred to as VZV-P; SEQ ID NO.46: An upstream primer designed for mumps virus, the sequence of which is 5'-atggtactcaaggactgtttgcc-3'; referred to as MuV-F; SEQ ID NO.47: A downstream primer designed for mumps virus, the sequence of which is 5'-cccaacaatattcgatgctagttcc-3'; abbreviated as MuV-R; SEQ ID NO.48: A fluorescent probe designed for mumps virus, the sequence of which is 5'-acaccacaaccacctgctttcaagataccg-3'; referred to as MuV-P; SEQ ID NO.49: An upstream primer designed for Streptococcus agalactiae, the sequence of the upstream primer is 5'-aagaagtattagcacaagagcaagc-3'; referred to as GBS-F; SEQ ID NO.50: A downstream primer designed for Streptococcus agalactiae, the sequence of which is 5'-tgttgttgactgactgactgacg-3'; referred to as GBS-R; SEQ ID NO.51: A fluorescent probe designed for Streptococcus agalactiae, the sequence of which is 5'-agtcaagcagcagccaatgaacaggtatcac-3'; referred to as GBS-P; SEQ ID NO.52: An upstream primer designed for Escherichia coli, the sequence of which is 5'-ggtgacggaagcgaatcagc-3'; referred to as E.coli-F; SEQ ID NO.53: A downstream primer designed for Escherichia coli, the sequence of which is 5'-gttgcggattaggcgtacagg-3'; referred to as E.coli-R; SEQ ID NO.54: A fluorescent probe designed for Escherichia coli, the sequence of which is 5'-acctctgcttggactgtttgctgacggc-3'; referred to as E.coli-P; SEQ ID NO.55: An upstream primer designed for Listeria, the sequence of which is 5'-actacaccaccttccgcaaatatac-3'; referred to as Listeria-F; SEQ ID NO.56: A downstream primer designed for Listeria, the sequence of which is 5'-gggtcataagcgttcattgtacttg-3'; referred to as Listeria-R; SEQ ID NO.57: A fluorescent probe designed for Listeria, the sequence of which is 5'-tggaagcaacacatctaacacatcaactggga-3'; referred to as Listeria-P; SEQ ID NO.58: An upstream primer designed for Neisseria meningitidis, the sequence of the upstream primer is 5'-ttatacacactgacaccaccttcac-3'; referred to as NME-F; SEQ ID NO.59: A downstream primer designed for Neisseria meningitidis, the sequence of the downstream primer is 5'-acaaataatcgctcaacgacattgc-3'; referred to as NME-R; SEQ ID NO.60: A fluorescent probe designed for Neisseria meningitidis, the sequence of which is 5'-ctatcatagccgccaccgccattcatgt-3'; referred to as NME-P; SEQ ID NO.61: An upstream primer designed for Streptococcus pneumoniae, the sequence of the upstream primer is 5'-ggtagctcctcagacagagtgg-3'; referred to as SPN-F; SEQ ID NO.62: A downstream primer designed for Streptococcus pneumoniae, the sequence of which is 5'-gtaaagcgactgccttcttgaatc-3'; referred to as SPN-R; SEQ ID NO.63: A fluorescent probe designed for Streptococcus pneumoniae, the sequence of which is 5'-agtcctctaccatatccaccttgcctgttaca-3'; referred to as SPN-P; SEQ ID NO.64: An upstream primer designed for pork tapeworm, the sequence of the upstream primer is 5'-caactgacaccgctgttctcc-3'; referred to as Taenia solium-F; SEQ ID NO.65: A downstream primer designed for Taenia solium, the sequence of which is 5'-ggtaaactcttagggcacgactg-3'; referred to as Taenia solium-R; SEQ ID NO.66: A fluorescent probe designed for pork tapeworm, the sequence of which is 5'-catcctacgcttgtgttgctaagtatggtccg-3'; referred to as Taenia solium-P; SEQ ID NO.67: An upstream primer designed for Schistosoma japonicum, the sequence of which is 5'-agacaggccgagatcagatacg-3'; referred to as Schistosoma-F; SEQ ID NO.68: A downstream primer designed for Schistosoma japonicum, the sequence of which is 5'-gtccagtaacctcaccacaatgc-3'; referred to as Schistosoma-R; SEQ ID NO.69: A fluorescent probe designed for Schistosoma japonicum, the sequence of which is 5'-acggaacacaaagcagagaagaaaccagccc-3'; abbreviated as Schistosoma-P; SEQ ID NO.70: An upstream primer designed for ribonuclease P, the sequence of the upstream primer is 5'-agatttggacctgcgagc-3'; referred to as RNase PF; SEQ ID NO.71: A downstream primer designed for ribonuclease P, the sequence of which is 5'-gagcggctgtctccacaagt-3'; referred to as RNase PR; SEQ ID NO.72: A fluorescent probe designed for ribonuclease P, the sequence of which is 5'-ttctgacctgaaggctctgcgcg-3'; referred to as RNase PP.

[0010] The fluorescent probe of the present invention has a 5' end labeled with a fluorescent group selected from any one of 6-FAM, ROX, CY5 and VIC, and a 3' end labeled with a quenching group selected from any one of BHQ1, BHQ2 or BHQ3, which can meet the needs of multi-channel and high-throughput detection.

[0011] The primer probe introduced into the present invention as an internal reference can monitor the entire process of collecting, transporting, extracting and amplifying the sample to be tested.

[0012] A kit for detecting brain infection pathogens, comprising a PCR reaction system, a positive control and a negative control; the PCR reaction system comprises a primer-probe combination having a nucleotide sequence as shown in SEQ ID NO.1-SEQ ID NO.72, a reaction buffer and an enzyme mixture.

[0013] The positive controls are CHIKV artificial synthetic plasmid, StLEV artificial synthetic plasmid, JEV artificial synthetic plasmid, TBEV artificial synthetic plasmid, EEEV artificial synthetic plasmid, WEEV artificial synthetic plasmid, WNV artificial synthetic plasmid, ECHO artificial synthetic plasmid, CVA6 artificial synthetic plasmid, CVA10 artificial synthetic plasmid, CVA16 artificial synthetic plasmid, CMV artificial synthetic plasmid, HSV1 artificial synthetic plasmid, HSV2 artificial synthetic plasmid, VZV artificial synthetic plasmid, MuV artificial synthetic plasmid, GBS artificial synthetic plasmid, E.coli artificial synthetic plasmid, Listeria artificial synthetic plasmid, NME artificial synthetic plasmid, SPN artificial synthetic plasmid, Taenia A plasmid mixture of solium artificial synthetic plasmid, Schistosoma artificial synthetic plasmid and internal reference artificial synthetic plasmid, the concentration ratio of the above artificial synthetic plasmids is 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1.

[0014] The negative control is TE Buffer.

[0015] The internal reference artificial synthetic plasmid is a plasmid into which an RNase P gene sequence fragment is inserted.

[0016] The method for preparing a kit for detecting pathogens of brain infection comprises the following steps: preparing a PCR reaction system, preparing a positive control and a negative control, assembling a detection kit, and constructing a reaction program.

[0017] The reaction program of the construction includes: the reaction program includes reverse transcription at 50°C for 10 minutes; pre-denaturation at 95°C for 5 minutes; amplification reaction: reaction at 95°C for 15 seconds, reaction at 60°C for 30 seconds, and 40 cycles; cycle reaction: reaction at 9°C for 3 minutes, reaction at 40°C for 3 minutes; melting program: heating rate of 45°C-80°C at 0.05°C / s.

[0018] In the amplification reaction step, fluorescence is detected at 60°C; In the melting program step, fluorescence is detected at 45°C-80°C, and fluorescence signals of FAM, ROX, CY5, and VIC channels are collected.

[0019] The beneficial effects of the present invention are:

[0020] The present invention solves the technical problem that the qPCR detection platform in the prior art is difficult to detect multiple pathogens simultaneously in the same reaction tube. By using the probe melting curve technology, by screening suitable primer probes and optimizing the reaction system and reaction procedure, a kit for detecting pathogens related to brain infection and its application are provided, which can detect 20 related pathogens that can cause brain infection under four fluorescent channels, and determine 23 different pathogens and serotypes by different Tm values. The present invention has simple operation steps, short detection cycle, high efficiency, low cost, accurate results, and avoids false positive phenomenon. In addition, the detection has high sensitivity and strong specificity, and realizes the possibility of multi-channel detection in a single tube system. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Melting curves of CHIKV, StLEV, JEV, TBEV, EEEV, and WEEV (FAM channel); Figure 2 Melting curves of WNV, ECHO, CVA6, CVA10, CVA16, and CMV (ROX channel); Figure 3 Melting curves of HSV1, HSV2, VZV, MuV, GBS, and E.coli (Cy5 channel); Figure 4 Melting curves of Listeria, NME, SPN, Taenia solium, Schistosoma, and internal reference (VIC channel). DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the present invention, those skilled in the art can make various technical changes or modifications to the present invention through technical attempts, and these equivalent forms generally fall within the scope defined by the appended claims of the present application.

[0023] Example 1 A primer combination for detecting brain infection pathogens

[0024] According to the NCBI GeneBank database, the gene sequences of CHIKV, StLEV, JEV, TBEV, EEEV, WEEV, WNV, ECHO, CVA6, CVA10, CVA16, CMV, HSV1, HSV2, VZV, MuV, GBS, E. coli, Listeria, NME, SPN, Taeniasolium, Schistosoma and human RNase P were obtained, the gene sequences were compared, and the following primers and probes were designed: SEQ ID NO.1: an upstream primer designed for Chikungunya virus, the sequence of the upstream primer is 5'-ccaacgctatcgcttgattacatc-3'; referred to as CHIKV-F; SEQ ID NO.2: A downstream primer designed for Chikungunya virus, the sequence of which is 5'-atgggtagacgccggtgaag-3'; referred to as CHIKV-R; SEQ ID NO.3: A fluorescent probe designed for Chikungunya virus, the sequence of which is 5'-tgcgaatacaaaaccgtcatcccgtctccg-3'; referred to as CHIKV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.4: An upstream primer designed for St. Louis encephalitis virus, the sequence of which is 5'-aaccacccttcggcgactc-3'; StLEV-F for short; SEQ ID NO.5: A downstream primer designed for St. Louis encephalitis virus, the sequence of which is 5'-gccttcccaatgctgcttcc-3'; referred to as StLEV-R; SEQ ID NO.6: A fluorescent probe designed for St. Louis encephalitis virus, the sequence of which is 5'-tatcgtcgtcggaagaggcaccaccca-3'; StLEV-P for short; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.7: An upstream primer designed for Japanese encephalitis virus, the sequence of which is 5'-tgacaacagaaggtgagaaagcc-3'; referred to as JEV-F; SEQ ID NO.8: A downstream primer designed for Japanese encephalitis virus, the sequence of which is 5'-gtggcctgacgttggtctttc-3'; referred to as JEV-R; SEQ ID NO.9: A fluorescent probe designed for Japanese encephalitis virus, the sequence of which is 5'-acttccagtgagcagggacctacttccgag-3'; referred to as JEV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.10: An upstream primer designed for forest encephalitis virus, the sequence of which is 5'-ctttcggacagcagagagtgttc-3'; referred to as TBEV-F; SEQ ID NO.11: A downstream primer designed for forest encephalitis virus, the sequence of which is 5'-gccagtcgttccaaaatccaatc-3'; referred to as TBEV-R; SEQ ID NO.12: A fluorescent probe designed for forest encephalitis virus, the sequence of which is 5'-agttgacccaaaggcacaggaacctcagc-3'; referred to as TBEV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.13: An upstream primer designed for Eastern Equine Encephalitis Virus of the United States, the sequence of the upstream primer is 5'-gtgtcaggtgtttacaggtgtttac-3'; referred to as EEEV-F; SEQ ID NO.14: A downstream primer designed for Eastern Equine Encephalitis Virus of the United States, the sequence of the downstream primer is 5'-catggtcgatagagcactcttcc-3'; referred to as EEEV-R; SEQ ID NO.15: A fluorescent probe designed for Eastern Equine Encephalitis Virus of the United States, the sequence of the fluorescent probe is 5'-cgctctacatacgcctcgctcatctggg-3'; referred to as EEEV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ1; SEQ ID NO.16: An upstream primer designed for Western equine encephalitis virus of the United States, the sequence of the upstream primer is 5'-ccaccgccaaagaagaagaagag-3'; referred to as WEEV-F; SEQ ID NO.17: A downstream primer designed for Western Equine Encephalitis Virus of the United States, the sequence of the downstream primer is 5'-cgctgtcgtttccctggtttag-3'; referred to as WEEV-R; SEQ ID NO.18: A fluorescent probe designed for Western Equine Encephalitis Virus of the United States, the sequence of which is 5'-cttgcgtttcgtcttcttggcttgttgcttct-3'; referred to as WEEV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.19: An upstream primer designed for West Nile virus, the sequence of the upstream primer is 5'-ggaggaacagagagacgttaatgg-3'; abbreviated as WNV-F; SEQ ID NO.20: A downstream primer designed for West Nile virus, the sequence of which is 5'-ccctcttgtgagcccaatgc-3'; abbreviated as WNV-R; SEQ ID NO.21: A fluorescent probe designed for West Nile virus, the sequence of which is 5'-tgaggaaccacacgccacgaagcagtc-3'; referred to as WNV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ2; SEQ ID NO.22: An upstream primer designed for echovirus, the sequence of which is 5'-gcgtccctgaagcagaaatgg-3'; referred to as ECHO-F; SEQ ID NO.23: A downstream primer designed for echovirus, the sequence of which is 5'-cgaatccgaagtgaacacatgagg-3'; referred to as ECHO-R; SEQ ID NO.24: A fluorescent probe designed for echovirus, the sequence of which is 5'-tcctcctcggtggtcaactctgtggca-3'; referred to as ECHO-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; SEQ ID NO.25: An upstream primer designed for Coxsackievirus group A type 6, the sequence of the upstream primer is 5'-gctttgatgccgagttcac-3'; abbreviated as CVA6-F; SEQ ID NO.26: A downstream primer designed for Coxsackievirus group A type 6, the sequence of the downstream primer is 5'-gtctgccattgatatgatttcc-3'; abbreviated as CVA6-R; SEQ ID NO.27: A fluorescent probe designed for Coxsackievirus group A type 6, the sequence of which is 5'-ccaacctcaatgacagcacgacgc-3'; referred to as CVA6-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ2; SEQ ID NO.28: An upstream primer designed for Coxsackievirus group A type 10, the sequence of the upstream primer is 5'-ttgtgtggttaacagaaatggg-3'; abbreviated as CVA10-F; SEQ ID NO.29: A downstream primer designed for Coxsackievirus A group 10, the sequence of the downstream primer is 5'-ctgcgttgaacctcatgtatg-3'; referred to as CVA10-R; SEQ ID NO.30: A fluorescent probe designed for Coxsackievirus A group 10, the sequence of which is 5'-aaccactatcaaccacttcttctcccgc-3'; referred to as CVA10-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ2; SEQ ID NO.31: An upstream primer designed for Coxsackievirus group A type 16, the sequence of the upstream primer is 5'-acacaagagacagccattgg-3'; abbreviated as CVA16-F; SEQ ID NO.32: A downstream primer designed for Coxsackievirus group A type 16, the sequence of the downstream primer is 5'-tcagcatcaaagcgcatg-3'; referred to as CVA16-R; SEQ ID NO.33: A fluorescent probe designed for Coxsackievirus A group 16, the sequence of which is 5'-aataactcgcatttccgccgcag-3'; referred to as CVA16-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ2; SEQ ID NO.34: An upstream primer designed for cytomegalovirus, the sequence of which is 5'-ttccccgaaggcaccagatg-3'; referred to as CMV-F; SEQ ID NO.35: A downstream primer designed for cytomegalovirus, the sequence of which is 5'-gggacctattcgttttcacacctac-3'; referred to as CMV-R; SEQ ID NO.36: A fluorescent probe designed for cytomegalovirus, the sequence of which is 5'-acgccgtgctcttcttcgactcgcc-3'; referred to as CMV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; SEQ ID NO.37: An upstream primer designed for herpes simplex virus type I, the sequence of the upstream primer is 5'-tggagcaccgagccaagg-3'; referred to as HSV1-F; SEQ ID NO.38: A downstream primer designed for herpes simplex virus type I, the sequence of the downstream primer is 5'-cgatgctgtccaccgtcac-3'; referred to as HSV1-R; SEQ ID NO.39: A fluorescent probe designed for herpes simplex virus type I, the sequence of the fluorescent probe is 5'-ctcctgtaagtacgccctcccgctgc-3'; referred to as HSV1-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ3; SEQ ID NO.40: An upstream primer designed for herpes simplex virus type II, the sequence of the upstream primer is 5'-caacttggcctcgtcaaaccc-3'; referred to as HSV2-F; SEQ ID NO.41: A downstream primer designed for herpes simplex virus type II, the sequence of the downstream primer is 5'-tgcaacgcaatcccatgaagg-3'; referred to as HSV2-R; SEQ ID NO.42: A fluorescent probe designed for herpes simplex virus type II, the sequence of which is 5'-ccgctcaccaccaaggaactcaagacttc-3'; referred to as HSV2-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ3; SEQ ID NO.43: An upstream primer designed for varicella-zoster virus, the sequence of the upstream primer is 5'-ctctggtgaagccgcacttac-3'; referred to as VZV-F; SEQ ID NO.44: A downstream primer designed for varicella-zoster virus, the sequence of which is 5'-cgaatgttggaaactgttggtcttc-3'; referred to as VZV-R; SEQ ID NO.45: A fluorescent probe designed for varicella-zoster virus, the sequence of which is 5'-ccgacccactctcccaaccctaacagc-3'; referred to as VZV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ3; SEQ ID NO.46: An upstream primer designed for mumps virus, the sequence of which is 5'-atggtactcaaggactgtttgcc-3'; referred to as MuV-F; SEQ ID NO.47: A downstream primer designed for mumps virus, the sequence of which is 5'-cccaacaatattcgatgctagttcc-3'; abbreviated as MuV-R; SEQ ID NO.48: A fluorescent probe designed for mumps virus, the sequence of which is 5'-acaccacaaccacctgctttcaagataccg-3'; referred to as MuV-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ3; SEQ ID NO.49: An upstream primer designed for Streptococcus agalactiae, the sequence of the upstream primer is 5'-aagaagtattagcacaagagcaagc-3'; referred to as GBS-F; SEQ ID NO.50: A downstream primer designed for Streptococcus agalactiae, the sequence of which is 5'-tgttgttgactgactgactgacg-3'; referred to as GBS-R; SEQ ID NO.51: A fluorescent probe designed for Streptococcus agalactiae, the sequence of which is 5'-agtcaagcagcagccaatgaacaggtatcac-3'; referred to as GBS-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ3; SEQ ID NO.52: An upstream primer designed for Escherichia coli, the sequence of which is 5'-ggtgacggaagcgaatcagc-3'; referred to as E.coli-F; SEQ ID NO.53: A downstream primer designed for Escherichia coli, the sequence of which is 5'-gttgcggattaggcgtacagg-3'; referred to as E.coli-R; SEQ ID NO.54: A fluorescent probe designed for Escherichia coli, the sequence of which is 5'-acctctgcttggactgtttgctgacggc-3'; referred to as E.coli-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ3; SEQ ID NO.55: An upstream primer designed for Listeria, the sequence of which is 5'-actacaccaccttccgcaaatatac-3'; referred to as Listeria-F; SEQ ID NO.56: A downstream primer designed for Listeria, the sequence of which is 5'-gggtcataagcgttcattgtacttg-3'; referred to as Listeria-R; SEQ ID NO.57: A fluorescent probe designed for Listeria, the sequence of which is 5'-tggaagcaacacatctaacacatcaactggga-3'; referred to as Listeria-P; the 5' end of the fluorescent probe is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.58: An upstream primer designed for Neisseria meningitidis, the sequence of the upstream primer is 5'-ttatacacactgacaccaccttcac-3'; referred to as NME-F; SEQ ID NO.59: A downstream primer designed for Neisseria meningitidis, the sequence of the downstream primer is 5'-acaaataatcgctcaacgacattgc-3'; referred to as NME-R; SEQ ID NO.60: A fluorescent probe designed for Neisseria meningitidis, the sequence of which is 5'-ctatcatagccgccaccgccattcatgt-3'; referred to as NME-P; the 5' end of the fluorescent probe is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.61: An upstream primer designed for Streptococcus pneumoniae, the sequence of the upstream primer is 5'-ggtagctcctcagacagagtgg-3'; referred to as SPN-F; SEQ ID NO.62: A downstream primer designed for Streptococcus pneumoniae, the sequence of which is 5'-gtaaagcgactgccttcttgaatc-3'; referred to as SPN-R; SEQ ID NO.63: A fluorescent probe designed for Streptococcus pneumoniae, the sequence of which is 5'-agtcctctaccatatccaccttgcctgttaca-3'; referred to as SPN-P; the 5' end of the fluorescent probe is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.64: An upstream primer designed for pork tapeworm, the sequence of the upstream primer is 5'-caactgacaccgctgttctcc-3'; referred to as Taenia solium-F; SEQ ID NO.65: A downstream primer designed for Taenia solium, the sequence of which is 5'-ggtaaactcttagggcacgactg-3'; referred to as Taenia solium-R; SEQ ID NO.66: A fluorescent probe designed for pork tapeworm, the sequence of which is 5'-catcctacgcttgtgttgctaagtatggtccg-3'; referred to as Taenia solium-P; the 5' end of the fluorescent probe is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group, and the quenching group is selected from BHQ1; SEQ ID NO.67: An upstream primer designed for Schistosoma japonicum, the sequence of which is 5'-agacaggccgagatcagatacg-3'; referred to as Schistosoma-F; SEQ ID NO.68: A downstream primer designed for Schistosoma japonicum, the sequence of which is 5'-gtccagtaacctcaccacaatgc-3'; referred to as Schistosoma-R; SEQ ID NO.69: A fluorescent probe designed for Schistosoma japonicum, the sequence of which is 5'-acggaacacaaagcagagaagaaaccagccc-3'; referred to as Schistosoma-P; the 5' end of the fluorescent probe is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.70: An upstream primer designed for ribonuclease P, the sequence of the upstream primer is 5'-agatttggacctgcgagc-3'; referred to as RNase PF; SEQ ID NO.71: A downstream primer designed for ribonuclease P, the sequence of which is 5'-gagcggctgtctccacaagt-3'; referred to as RNase PR; SEQ ID NO.72: A fluorescent probe designed for ribonuclease P, the sequence of which is 5'-ttctgacctgaaggctctgcgcg-3'; referred to as RNase PP; the 5' end of the fluorescent probe is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group BHQ1; The above primers and probe plasmids were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0025] Example 2 A method for preparing a kit for detecting pathogens of brain infection

[0026] Step 1: Prepare PCR reaction system The primer combination provided in Example 1 was used to prepare reagents, and a PCR reaction system for 100 tests was prepared according to Table 1.

[0027] Table 1 Components and dosage of PCR reaction system

[0028] 4×HU ® MP Buffer is a reaction buffer containing Mg 2+ , dNTP and dUTP, etc. The reaction buffer and primer probe are prepared in the same tube to form the PCR reaction solution. HU® Enzyme Mix (UDG plus) is an enzyme mixture, which contains reverse transcriptase, RNase inhibitor, Taq DNA polymerase and UDG enzyme. The enzyme mixture is separately packaged in a tube. The PCR reaction solution and the enzyme mixture together constitute the PCR reaction system of the kit. The enzyme mixture was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0029] Step 2: Prepare positive and negative controls Positive control: CHIKV target gene sequence, StLEV target gene sequence, JEV target gene sequence, TBEV target gene sequence, EEEV target gene sequence, WEEV target gene sequence, WNV target gene sequence, ECHO target gene sequence, CVA6 target gene sequence, CVA10 target gene sequence, CVA16 target gene sequence, CMV target gene sequence, HSV1 target gene sequence, HSV2 target gene sequence, VZV target gene sequence, MuV target gene sequence, GBS target gene sequence, E.coli target gene sequence, Listeria target gene sequence, NME target gene sequence, SPN target gene sequence, Taenia solium target gene sequence, Schistosoma target gene sequence, RNase The P gene sequences were inserted into the pUC57 vector plasmid respectively, and through expanded culture, identification and plasmid extraction, CHIKV artificial synthetic plasmid, StLEV artificial synthetic plasmid, JEV artificial synthetic plasmid, TBEV artificial synthetic plasmid, EEEV artificial synthetic plasmid, WEEV artificial synthetic plasmid, WNV artificial synthetic plasmid, ECHO artificial synthetic plasmid, CVA6 artificial synthetic plasmid, CVA10 artificial synthetic plasmid, CVA16 artificial synthetic plasmid, CMV artificial synthetic plasmid, HSV1 artificial synthetic plasmid, HSV2 artificial synthetic plasmid, VZV artificial synthetic plasmid, MuV artificial synthetic plasmid, GBS artificial synthetic plasmid, E.coli artificial synthetic plasmid, Listeria artificial synthetic plasmid, NME artificial synthetic plasmid, SPN artificial synthetic plasmid, Taenia solium artificial synthetic plasmid, Schistosoma artificial synthetic plasmid and internal reference artificial synthetic plasmid were obtained.

[0030] The above synthetic plasmids were quantified to 2.4×10 7 copies / mL, and mixed them in a volume ratio of 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1 to make a positive control; The negative control was TE Buffer.

[0031] Step 3: Assemble into a test kit The kit includes a PCR reaction system, a positive control and a negative control. The positive control and the negative control are packaged in 500 μL tubes each. The kit is stored in a -20°C refrigerator.

[0032] Step 4: Build the reaction program The reaction procedure is shown in Table 2 below: Table 2 Reaction procedures used in the present invention

[0033] The reaction program included reverse transcription at 50°C for 10 min; pre-denaturation at 95°C for 5 min; amplification reaction: 95°C for 15 s, 60°C for 30 s, and 40 cycles; cycling reaction: 9°C for 3 min, 40°C for 3 min; melting program: 45°C-80°C at a heating rate of 0.05°C / s.

[0034] During the amplification reaction step, fluorescence was detected at 60°C; during the melting program step, fluorescence was detected at 45°C-80°C, and fluorescence signals of FAM, ROX, CY5, and VIC channels were collected.

[0035] Example 3: Test kit sensitivity performance verification 1. Template preparation The CHIKV target gene sequence, StLEV target gene sequence, JEV target gene sequence, TBEV target gene sequence, EEEV target gene sequence, WEEV target gene sequence, WNV target gene sequence, ECHO target gene sequence, CVA6 target gene sequence, CVA10 target gene sequence, CVA16 target gene sequence, CMV target gene sequence, HSV1 target gene sequence, HSV2 target gene sequence, VZV target gene sequence, MuV target gene sequence, GBS target gene sequence, E. coli target gene sequence, Listeria target gene sequence, NME target gene sequence, SPN target gene sequence, Taenia solium target gene sequence, and Schistosoma target gene sequence were respectively inserted into pUC 57 vector plasmids were obtained through expanded culture, identification and plasmid extraction, including CHIKV artificial synthetic plasmid, StLEV artificial synthetic plasmid, JEV artificial synthetic plasmid, TBEV artificial synthetic plasmid, EEEV artificial synthetic plasmid, WEEV artificial synthetic plasmid, WNV artificial synthetic plasmid, ECHO artificial synthetic plasmid, CVA6 artificial synthetic plasmid, CVA10 artificial synthetic plasmid, CVA16 artificial synthetic plasmid, CMV artificial synthetic plasmid, HSV1 artificial synthetic plasmid, HSV2 artificial synthetic plasmid, VZV artificial synthetic plasmid, MuV artificial synthetic plasmid, GBS artificial synthetic plasmid, E.coli artificial synthetic plasmid, Listeria artificial synthetic plasmid, NME artificial synthetic plasmid, SPN artificial synthetic plasmid, Taenia solium artificial synthetic plasmid and Schistosoma artificial synthetic plasmid.

[0036] The above synthetic plasmids were quantified by Qubit and diluted to 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×104 copies / mL, 1×10 3 copies / mL.

[0037] 2. Probe melting curve method to detect pathogen plasmids The kit assembled in Example 2 was taken out of the -20°C refrigerator and equilibrated to room temperature. After being fully shaken and briefly centrifuged, N×18 μL of PCR reaction solution and N×2 μL of enzyme mixture were added to 1.5 mL centrifuge tubes. The centrifuge tubes were fully shaken and mixed, and then centrifuged for ten seconds. The PCR reaction system was divided into eight tubes with 20 μL per reaction, and the concentration of 1×10 3 Add 5 μL of each of the plasmid, positive control and negative control with 5 copies / mL, cap the tube tightly and centrifuge instantly, put the PCR reaction tube on the Macrostone 96S fluorescent PCR amplifier, set the name of each sample, and perform amplification detection according to the reaction program in Table 2.

[0038] 3. Analysis of amplification results The melting curve test results of different pathogens and internal reference synthetic plasmids are shown in Table 3 and Figure 1-Figure 4 shown.

[0039] The Tm values ​​of the melting curve method for each pathogen are shown in Table 3.

[0040] Table 3 Tm values ​​of the melting curve method for each pathogen

[0041] Result analysis: like Figure 1 As shown in the figure, the Tm value of CHIKV is 55.2℃, the Tm value of StLEV is 76.8℃, the Tm value of JEV is 59.2℃, the Tm value of TBEV is 68.8℃, the Tm value of EEEV is 71.6℃, and the Tm value of WEEV is 62.8℃. The detection sensitivity of the six pathogens is 1×10 3 copies / mL.

[0042] like Figure 2 As shown in the figure, the Tm value of WNV is 71.6℃, the Tm value of ECHO is 55.6℃, the Tm value of CVA6 is 59.2℃, the Tm value of CVA10 is 76.8℃, the Tm value of CVA16 is 67.6℃, and the Tm value of CMV is 64.8℃. The detection sensitivity of the six pathogens is 1×10 3 copies / mL.

[0043] like Figure 3As shown in the figure, the Tm value of HSV1 is 72.4℃, the Tm value of HSV2 is 56℃, the Tm value of VZV is 64℃, the Tm value of MuV is 77.2, the Tm value of GBS is 67.2℃, and the Tm value of E.coli is 60℃. The detection sensitivity of the six pathogens is 1×10 3 copies / mL.

[0044] like Figure 4 As shown in the figure, the Tm value of Listeria is 71.6℃, the Tm value of NME is 61.2℃, the Tm value of SPN is 58℃, the Tm value of Taenia solium is 76℃, the Tm value of Schistosoma is 55.2℃, and the Tm value of the internal reference artificial synthetic plasmid is 66.4℃. The detection sensitivity of the five pathogens is 1×10 3 copies / mL.

[0045] Example 4 Kit Specificity Detection Experiment 1. Template preparation The nucleic acid extraction or purification reagents of Qingdao Han Tang Biotechnology Co., Ltd. were used to extract the quality control products of Chikungunya virus, St. Louis encephalitis virus, Japanese encephalitis virus, forest encephalitis virus, Eastern American equine encephalitis virus, Western American equine encephalitis virus, West Nile virus, echovirus, coxsackievirus A group 6, coxsackievirus A group 10 and coxsackievirus A group 16, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, mumps virus, group B Streptococcus, Escherichia coli, Listeria, Neisseria meningitidis, Streptococcus pneumoniae, pork tapeworm, Schistosoma japonicum, as well as Bartonella, dengue virus, Lassa fever virus, Zika virus, human bocavirus, dengue virus, Lassa fever virus, Zika virus, Escherichia coli, Vibrio cholerae, Salmonella, Shigella, Vibrio parahaemolyticus, and Salmonella typhi, and 100 μL of nucleic acid was obtained for each.

[0046] 2. Probe melting curve method for detecting pathogen quality control products The nucleic acid of the quality control product was detected according to the method of Example 3.

[0047] 3. Results Analysis The test results are shown in Table 4. In addition to Chikungunya virus, St. Louis encephalitis virus, Japanese encephalitis virus, forest encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, West Nile virus, echovirus, coxsackievirus A group 6, coxsackievirus A group 10 and coxsackievirus A group 16, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, varicella-zoster virus, mumps virus, group B streptococcus, Escherichia coli, Except for Listeria, Neisseria meningitidis, Streptococcus pneumoniae, pork tapeworm, and Schistosoma japonicum, no melting peaks appeared for Bartonella, dengue virus, Lassa fever virus, Zika virus, human bocavirus, yellow fever virus, West Nile virus, norovirus, novel Bunyavirus, Rift Valley fever virus, rotavirus, Vibrio cholerae, Salmonella, Shigella, Vibrio parahaemolyticus, Salmonella typhi, and Candida albicans, indicating that the kit has good specificity and no cross-reaction with other pathogens.

[0048] Table 4 Specificity analysis verification results

[0049] Example 5: Application of the kit to the detection of actual samples 1. Template preparation A total of 56 CSF samples were collected clinically and numbered 1 to 56, including 3 CHIKV-infected CSF samples, 2 StLEV-infected CSF samples, 2 JEV-infected CSF samples, 3 TBEV-infected CSF samples, 3 EEEV-infected CSF samples, 2 WEEV-infected CSF samples, 3 WNV-infected CSF samples, 2 ECHO-infected CSF samples, 2 CV6A-infected CSF samples, and 2 CVA10-infected CSF samples. The cerebrospinal fluid samples were collected from 24 samples of Wuhan University, 3 samples of Wuhan University, 3 samples of Wuhan University, 2 samples of Wuhan University, 3 samples of Wuhan University, 2 samples of Wuhan University, 2 samples of Wuhan University, 2 samples of Wuhan University, 2 samples of Wuhan University, 2 samples of Wuhan University, 2 samples of Wuhan University, 2 samples of Wuhan University, and 2 samples of Wuhan University. The nucleic acid extraction or purification reagents of Qingdao Han Tang Biotechnology Co., Ltd. were used for extraction to obtain 100 μL of nucleic acid each.

[0050] 2. Fluorescence PCR detection The above actual sample was tested according to the method of Example 3.

[0051] 3. Results Analysis The test results of 56 clinical samples are shown in Table 5. All pathogen samples can be detected normally, and the Tm value of each pathogen sample is basically consistent with the Tm value of the channel corresponding to the positive control, which is consistent with the previous diagnosis results.

[0052] Table 5 Detection results of various pathogen samples

[0053] It should be noted that the above-described embodiments of the present invention do not limit the protection scope of the present invention. Any technical solution obtained by equivalent replacement or equivalent transformation should be included in the protection scope of the present invention.

Claims

1. A primer-probe combination for detecting pathogens of brain infection, characterized in that: The primer-probe composition comprises an upstream primer designed for Chikungunya virus, a downstream primer designed for Chikungunya virus, a fluorescent probe designed for Chikungunya virus, an upstream primer designed for St. Louis encephalitis virus, a downstream primer designed for St. Louis encephalitis virus, a fluorescent probe designed for St. Louis encephalitis virus, an upstream primer designed for Japanese encephalitis virus, a downstream primer designed for Japanese encephalitis virus, a fluorescent probe designed for Japanese encephalitis virus, an upstream primer designed for forest encephalitis virus, a downstream primer designed for forest encephalitis virus, a fluorescent probe designed for forest encephalitis virus, an upstream primer designed for Eastern American equine encephalitis virus, a downstream primer designed for Eastern American equine encephalitis virus, and a fluorescent probe designed for Eastern American equine encephalitis virus. Fluorescent probe designed for western equine encephalitis virus, upstream primer designed for western equine encephalitis virus, downstream primer designed for western equine encephalitis virus, fluorescent probe designed for western equine encephalitis virus, upstream primer designed for West Nile virus, downstream primer designed for West Nile virus, fluorescent probe designed for West Nile virus, upstream primer designed for echovirus, downstream primer designed for echovirus, fluorescent probe designed for echovirus, upstream primer designed for coxsackievirus group A 6, downstream primer designed for coxsackievirus group A 6, fluorescent probe designed for coxsackievirus group A 6, upstream primer designed for coxsackievirus group A 10, downstream primer designed for coxsackievirus group A 10 downstream primers designed for Coxsackievirus A group 10, upstream primers designed for Coxsackievirus A group 16, downstream primers designed for Coxsackievirus A group 16, fluorescent probe designed for Coxsackievirus A group 16, upstream primers designed for cytomegalovirus, downstream primers designed for cytomegalovirus, fluorescent probe designed for cytomegalovirus, upstream primers designed for herpes simplex virus type I, downstream primers designed for herpes simplex virus type I, fluorescent probe designed for herpes simplex virus type I, upstream primers designed for herpes simplex virus type II, downstream primers designed for herpes simplex virus type II, fluorescent probe designed for herpes simplex virus type II, upstream primers designed for varicella-zoster virus Upstream primers designed for herpes virus, downstream primers designed for varicella-zoster virus, fluorescent probes designed for varicella-zoster virus, upstream primers designed for mumps virus, downstream primers designed for mumps virus, fluorescent probes designed for mumps virus, upstream primers designed for Streptococcus agalactiae, downstream primers designed for Streptococcus agalactiae, fluorescent probes designed for Streptococcus agalactiae, upstream primers designed for Escherichia coli, downstream primers designed for Escherichia coli, fluorescent probes designed for Escherichia coli, upstream primers designed for Listeria, downstream primers designed for Listeria, fluorescent probes designed for Listeria, upstream primers designed for Neisseria meningitidis,Downstream primers designed for Neisseria meningitidis, fluorescent probes designed for Neisseria meningitidis, upstream primers designed for Streptococcus pneumoniae, downstream primers designed for Streptococcus pneumoniae, fluorescent probes designed for Streptococcus pneumoniae, upstream primers designed for pork tapeworm, downstream primers designed for pork tapeworm, fluorescent probes designed for pork tapeworm, upstream primers designed for schistosome, downstream primers designed for schistosome, fluorescent probes designed for schistosome, upstream primers designed for ribonuclease P, downstream primers designed for ribonuclease P, fluorescent probes designed for ribonuclease P; The upstream primer designed for Chikungunya virus: the sequence is shown in SEQ ID NO.1; The downstream primer designed for Chikungunya virus: the sequence is shown in SEQ ID NO.2; The fluorescent probe designed for Chikungunya virus: the sequence is shown in SEQ ID NO.3; The upstream primer designed for St. Louis encephalitis virus: the sequence is shown in SEQ ID NO.4; The downstream primer designed for St. Louis encephalitis virus: the sequence is shown in SEQ ID NO.5; The fluorescent probe designed for St. Louis encephalitis virus: the sequence is shown in SEQ ID NO.6; The upstream primer designed for Japanese encephalitis virus: the sequence is shown in SEQ ID NO.7; The downstream primer designed for Japanese encephalitis virus: the sequence is shown in SEQ ID NO.8; The fluorescent probe designed for Japanese encephalitis virus: the sequence is shown in SEQ ID NO.9; The upstream primer designed for forest encephalitis virus: the sequence is shown in SEQ ID NO.10; The downstream primer designed for forest encephalitis virus: the sequence is shown in SEQ ID NO.11; The fluorescent probe designed for the forest encephalitis virus: the sequence is shown in SEQ ID NO.12; The upstream primer designed for Eastern Equine Encephalitis Virus of the United States: the sequence is shown in SEQ ID NO.13; The downstream primer designed for Eastern American equine encephalitis virus: the sequence is shown in SEQ ID NO.14; The fluorescent probe designed for Eastern Equine Encephalitis Virus of the United States: the sequence is shown in SEQ ID NO.15; The upstream primer designed for Western American equine encephalitis virus: the sequence is shown in SEQ ID NO.16; The downstream primer designed for Western American equine encephalitis virus: the sequence is shown in SEQ ID NO.17; The fluorescent probe designed for Western American equine encephalitis virus: the sequence is shown in SEQ ID NO.18; The upstream primer designed for West Nile virus: the sequence is shown in SEQ ID NO.19; The downstream primer designed for West Nile virus: the sequence is shown in SEQ ID NO.20; The fluorescent probe designed for West Nile virus: the sequence is shown in SEQ ID NO.21; The upstream primer designed for echovirus: the sequence is shown in SEQ ID NO.22; The downstream primer designed for echovirus: the sequence is shown in SEQ ID NO.23; The fluorescent probe designed for echovirus: the sequence is shown in SEQ ID NO.24; The upstream primer designed for Coxsackievirus A group 6: the sequence is shown in SEQ ID NO.25; The downstream primer designed for Coxsackievirus A group 6: the sequence is shown in SEQ ID NO.26; The fluorescent probe designed for Coxsackievirus A group 6: the sequence is shown in SEQ ID NO.27; The upstream primer designed for Coxsackievirus A group 10: the sequence is shown in SEQ ID NO.28; The downstream primer designed for Coxsackievirus A group 10: the sequence is shown in SEQ ID NO.29; The fluorescent probe designed for Coxsackievirus A group 10: the sequence is shown in SEQ ID NO.30; The upstream primer designed for Coxsackievirus A group 16: the sequence is shown in SEQ ID NO.31; The downstream primer designed for Coxsackievirus A group 16: the sequence is shown in SEQ ID NO.32; The fluorescent probe designed for Coxsackievirus A group 16: the sequence is shown in SEQ ID NO.33; The upstream primer designed for cytomegalovirus: the sequence is shown in SEQ ID NO.34; The downstream primer designed for cytomegalovirus: the sequence is shown in SEQ ID NO.35; The fluorescent probe designed for cytomegalovirus: the sequence is shown in SEQ ID NO.36; The upstream primer designed for herpes simplex virus type I: the sequence is shown in SEQ ID NO.37; The downstream primer designed for herpes simplex virus type I: the sequence is shown in SEQ ID NO.38; The fluorescent probe designed for herpes simplex virus type I: the sequence is shown in SEQ ID NO.39; The upstream primer designed for herpes simplex virus type II: the sequence is shown in SEQ ID NO.40; The downstream primer designed for herpes simplex virus type II: the sequence is shown in SEQ ID NO.41; The fluorescent probe designed for herpes simplex virus type II: the sequence is shown in SEQ ID NO.42; The upstream primer designed for varicella-zoster virus: the sequence is shown in SEQ ID NO.43; The downstream primer designed for varicella-zoster virus: the sequence is shown in SEQ ID NO. 44; The fluorescent probe designed for varicella-zoster virus: the sequence is shown in SEQ ID NO.45; The upstream primer designed for mumps virus: the sequence is shown in SEQ ID NO.46; The downstream primer designed for mumps virus: the sequence is shown in SEQ ID NO.47; The fluorescent probe designed for mumps virus: the sequence is shown in SEQ ID NO.48; The upstream primer designed for Streptococcus agalactiae: the sequence is shown in SEQ ID NO.49; The downstream primer designed for Streptococcus agalactiae: the sequence is shown in SEQ ID NO.50; The fluorescent probe designed for Streptococcus agalactiae: the sequence is shown in SEQ ID NO.51; The upstream primer designed for Escherichia coli has a sequence as shown in SEQ ID NO.52; The downstream primer designed for Escherichia coli: the sequence is shown in SEQ ID NO.53; The fluorescent probe designed for Escherichia coli: the sequence is shown in SEQ ID NO.54; The upstream primer designed for Listeria: the sequence is shown in SEQ ID NO.55; The downstream primer designed for Listeria: the sequence is shown in SEQ ID NO.56; The fluorescent probe designed for Listeria: the sequence is shown in SEQ ID NO.57; The upstream primer designed for Neisseria meningitidis: the sequence is shown in SEQ ID NO.58; The downstream primer designed for Neisseria meningitidis: the sequence is shown in SEQ ID NO.59; The fluorescent probe designed for Neisseria meningitidis: the sequence is shown in SEQ ID NO.60; The upstream primer designed for Streptococcus pneumoniae: the sequence is shown in SEQ ID NO.61; The downstream primer designed for Streptococcus pneumoniae: the sequence is shown in SEQ ID NO.62; The fluorescent probe designed for Streptococcus pneumoniae: the sequence is shown in SEQ ID NO.63; The upstream primer designed for pork tapeworm: the sequence is shown in SEQ ID NO.64; The downstream primer designed for pork tapeworm: the sequence is shown in SEQ ID NO.65; The fluorescent probe designed for pork tapeworm: the sequence is shown in SEQ ID NO.66; The upstream primer designed for Schistosoma japonicum: the sequence is shown in SEQ ID NO.67; The downstream primer designed for Schistosoma japonicum: the sequence is shown in SEQ ID NO.68; The fluorescent probe designed for Schistosoma japonicum: the sequence is shown in SEQ ID NO.69; The upstream primer designed for ribonuclease P: the sequence is shown in SEQ ID NO.70; The downstream primer designed for ribonuclease P: the sequence is shown in SEQ ID NO.71; The fluorescent probe designed for ribonuclease P: the sequence is shown in SEQ ID NO.

72.

2. A primer-probe combination for detecting pathogens of brain infection according to claim 1, characterized in that: The 5' end of the fluorescent probe is labeled with a fluorescent group selected from any one of 6-FAM, ROX, CY5 and VIC, and the 3' end is labeled with a quenching group selected from any one of BHQ1, BHQ2 or BHQ3.

3. A kit for detecting pathogens of brain infection, characterized in that: It comprises a PCR reaction system, a positive control and a negative control; the PCR reaction system comprises a primer probe combination having a nucleotide sequence as shown in SEQ ID NO.1-SEQ ID NO.72, a reaction buffer and an enzyme mixture.

4. A kit for detecting pathogens of brain infection according to claim 3, characterized in that: The positive controls are CHIKV artificial synthetic plasmid, StLEV artificial synthetic plasmid, JEV artificial synthetic plasmid, TBEV artificial synthetic plasmid, EEEV artificial synthetic plasmid, WEEV artificial synthetic plasmid, WNV artificial synthetic plasmid, ECHO artificial synthetic plasmid, CVA6 artificial synthetic plasmid, CVA10 artificial synthetic plasmid, CVA16 artificial synthetic plasmid, CMV artificial synthetic plasmid, HSV1 artificial synthetic plasmid, HSV2 artificial synthetic plasmid, VZV artificial synthetic plasmid, MuV artificial synthetic plasmid, GBS artificial synthetic plasmid, E.coli artificial synthetic plasmid, Listeria artificial synthetic plasmid, NME artificial synthetic plasmid, SPN artificial synthetic plasmid, Taenia A plasmid mixture of solium artificial synthetic plasmid, Schistosoma artificial synthetic plasmid and internal reference artificial synthetic plasmid, the concentration ratio of the above artificial synthetic plasmids is 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:

1.

5. A kit for detecting pathogens of brain infection according to claim 4, characterized in that: The negative control is TE Buffer.

6. A kit for detecting pathogens of brain infection according to claim 4, characterized in that: The internal reference artificial synthetic plasmid is a plasmid into which an RNase P gene sequence fragment is inserted.

7. A method for preparing a kit for detecting pathogens of brain infection, characterized in that: The following steps are involved: A PCR reaction system, a positive control and a negative control were prepared, a detection kit was assembled, and a reaction program was constructed. The reaction program included reverse transcription at 50°C for 10 minutes; pre-denaturation at 95°C for 5 minutes; amplification reaction: 95°C for 15 seconds, 60°C for 30 seconds, and 40 cycles; cycle reaction: 9°C for 3 minutes, 40°C for 3 minutes; melting program: 45°C-80°C with a heating rate of 0.05°C / s.

8. The method for preparing a kit for detecting pathogens of brain infection according to claim 7, characterized in that: In the amplification reaction step, fluorescence is detected at 60°C.

9. The method for preparing a kit for detecting pathogens of brain infection according to claim 7, characterized in that: In the melting program step, fluorescence is detected at 45°C-80°C, and fluorescence signals of FAM, ROX, CY5, and VIC channels are collected.

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